microorganisms

Article Identification of A Novel Arsenic Resistance Transposon Nested in A Mercury Resistance Transposon of Bacillus sp. MB24

Mei-Fang Chien 1 , Ying-Ning Ho 1,2, Hui-Erh Yang 3, Masaru Narita 4,5, Keisuke Miyauchi 4, Ginro Endo 4 and Chieh-Chen Huang 3,*

1 Graduate School of Environmental Studies, Tohoku University, Aramaki, Aoba-ku, 6-6-20 Aoba, Sendai 980-8579, Japan; [email protected] (M.-F.C.); [email protected] (Y.-N.H.) 2 Institute of Marine Biology and Center of Excellence for the Oceans, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 20224, Taiwan 3 Department of Life Sciences, National Chung Hsing University, 145 Xingda Road, Taichung 40227, Taiwan; [email protected] 4 Faculty of Engineering, Tohoku Gakuin University, 1-13-1 Chuo, Tagajo, Miyagi 985-8537, Japan; [email protected] (M.N.); [email protected] (K.M.); [email protected] (G.E.) 5 Tohoku Afforestation and Environmental Protection Ltd., 2-5-1 Honmachi, Aobaku, Sendai 980-0014, Japan * Correspondence: [email protected]; Tel.: +886-4-2284-0416



Received: 10 October 2019; Accepted: 12 November 2019; Published: 16 November 2019


Abstract: A novel TnMERI1-like transposon designated as TnMARS1 was identified from mercury resistant Bacilli isolated from Minamata Bay sediment. Two adjacent ars operon-like gene clusters, ars1 and ars2, flanked by a pair of 78-bp inverted repeat sequences, which resulted in a 13.8-kbp transposon-like fragment, were found to be sandwiched between two transposable genes of the TnMERI1-like transposon of a mercury resistant bacterium, Bacillus sp. MB24. The presence of a single transcription start site in each cluster determined by 50-RACE suggested that both are operons. Quantitative real time RT-PCR showed that the transcription of the arsR genes contained in each operon was induced by arsenite, while arsR2 responded to arsenite more sensitively and strikingly than arsR1 did. Further, arsenic resistance complementary experiments showed that the ars2 operon conferred arsenate and arsenite resistance to an arsB-knocked out Bacillus host, while the ars1 operon only raised arsenite resistance slightly. This transposon nested in TnMARS1 was designated as TnARS1. Multi-gene cluster blast against bacteria and Bacilli whole genome sequence databases suggested that TnMARS1 is the first case of a TnMERI1-like transposon combined with an arsenic resistance transposon. The findings of this study suggested that TnMERI1-like transposons could recruit other mobile elements into its genetic structure, and subsequently cause horizontal dissemination of both mercury and arsenic resistances among Bacilli in Minamata Bay.

Keywords: class II transposon; arsenic resistance operon; mercury resistance operon; genome mining; horizontal gene transfer

1. Introduction Horizontal gene transfer is a mechanism of giving-and-taking genetic elements between different species and genera, which is a strategy of organisms for survival and plays an important role in microbial evolution. The event of horizontal gene transfer has been confirmed by both laboratory and field studies, and is believed to be driven by environmental stresses, like hazardous pollutions [1]. Mercury and its related compounds are recognized as hazardous pollutants. Among these, the highly

Microorganisms 2019, 7, 566; doi:10.3390/microorganisms7110566 www.mdpi.com/journal/microorganisms Microorganisms 2019, 7, 566 2 of 11 toxic methylmercury chloride is widely known as it has been identified as a causative agent of Minamata disease. Though mercury compounds are generally toxic to all living organisms, mercury resistance genes (mer operon) were found in many genera of environmental bacteria. Several studies have described the identification and the possible mechanism of horizontal transfer of mer genes in both Gram-negative and Gram-positive bacteria [2,3]. The majority of mercury resistance transposons that have been studied are class II transposons, which are characterized by the presence of 35–48-bp terminal inverted repeats (IRs), transposase (tnpA) and resolvase (tnpR) genes, and a res-internal resolution site [4]. Bacillus megaterium MB1, an isolate from Minamata Bay, Japan, contains a broad-spectrum mercury resistance determinant encoded in a chromosomal class II transposon, TnMERI1 [4]. Besides B. megaterium MB1, 30 Bacillus strains were isolated from mercury contaminated Minamata Bay sediment and the genetic characteristics of the mer determinants were analyzed [5]. Eleven of the 30 Bacillus strains showed broad-spectrum mercury resistance and contained mer operons identical to that of Bacillus megaterium MB1 [4,6]. However, the localization of these mer operons in these Bacillus strains remained unclear. On the other hand, with the development of sequencing technology, the whole genome database continues to grow and has proven to be suitable in surveying specific gene clusters. By using genome mining approaches, we have found broad-spectrum mercury resistance gene clusters in not only mercury-polluted Minamata Bay sediment strains but also Bacillus sp. strains from water, food and animal sources [7]. On the other hand, arsenic is also recognized as one of the most toxic oxyanions in the natural environment known to cause severe contamination of soil-water systems that has resulted in “blackfoot disease” in endemic areas of some countries such as Bangladesh, India, and Taiwan. Like mercury, many microorganisms have evolved different arsenic detoxification pathways to cope with the widespread distribution of the said pollutant. One of the most characterized pathways is cytoplasmic arsenate reduction and arsenite extrusion [8,9]. Genes for arsenic detoxification were first discovered and characterized in Gram-negative bacteria [8,10]. These genes are often plasmid-encoded and are widespread in prokaryotes [11–15]. In thoroughly studied systems, the ars operon has been reported to contain five genes arsRDABC, as in Escherichia coli [15,16], or at least the three genes arsRBC, as in Staphylococcus aureus [17]. ArsR is an arsenite-responsive repressor that works in dimeric form and binds to the ars promoter [18]. ArsA and ArsB are components of an arsenite-transporter, where ArsA is the ATPase and ArsB is the transmembrane component of the complex. In microorganisms in which ArsA is absent, ArsB acts as a single-component transporter. ArsC is the arsenate reductase that converts arsenate As(V) to arsenite As(III) [19–23], which is subsequently pumped out of the cell through an energy-dependent efflux process [24]. More recently, new arrangements of arsenic-resistance genes have been discovered in a number of microorganisms, including Bacteria [19–23] and Archaea [25]. It is worth noticing that there is a low similarity of 16S rRNA among these microbes, which suggests horizontal transfer among these bacteria. However, there is no report about arsenic resistance mobile elements with mercury resistance ones. In the present study, Minamata Bay-isolated Bacillus containing broad-spectrum mercury resistance were used to investigate the localization of these mercury resistance transposons. A novel type of TnMERI1-like transposon, TnMARS1, was identified, and a transposon-like fragment, namely TnARS1, was found nested in TnMARS1. The second motivation of this study was to investigate the functions of components in this novel transposon. The results showed that TnARS1 contains a functional arsenic resistance operon, and that TnMARS1 is a newly isolated TnMERI1-like mercury resistance transposon nesting an arsenic resistance transposon. The diversity of TnMERI1-like transposon in the public Bacillus database was also investigated, and the result of genome mining showed that TnMARS1 is the first case in which an arsenic resistance transposon has been identified from a TnMERI1-like transposon. The present study also suggested that the dissemination of TnMERI1-like transposons might contribute to the recruitment of other mobile genetic elements into the Bacilli of Minamata Bay. Microorganisms 2019, 7, 566 3 of 11

2. Materials and Methods

2.1. Bacterial Strains, Plasmids, and Culture Conditions Eleven mercury resistant Bacillus strains (included Bacillus sp. MB24) isolated from our previous study were used [5]. Bacillus sp. MB24 was used in the 50-RACE experiment. Bacillus subtilis 168 [26] and its derivative with a knocked out arsB were used in the arsenic resistance assay. Plasmids pHYARS1, pHYARS2 and pHYARS3 were derivatives of the vector pHY300PLK (Takara Bio Inc., Shiga, Japan), where the arsR1-orf5, arsR2-orf12 and arsR1-orf12 regions of Bacillus sp. MB24 were cloned, respectively, into the BamHI and XbaI site of pHY300PLK. The bacterial cells were grown in Luria Broth (LB) medium at 37 ◦C with agitation. Tetracycline and chloramphenicol were added as required at the concentration of 25 µg/mL and 5 µg/mL, respectively.

2.2. PCR Amplification of Intact Transposon Regions The intact mercury resistance transposon regions of the 11 Bacillus strains were amplified by using polymerase chain reaction (PCR) with an IR-targeted single PCR primer [4]. The primer sequence is shown in Table S1, and the conditions of PCR amplification were described in our previous studies [4,27]. The amplicons were digested with restriction endonucleases BglII, HindIII, NcoI and SmaI (Takara Bio Inc.). The digested DNA fragments were electrophoresed on agarose gels and the gels were stained with ethidium bromide (0.5 µg/mL) and subsequently photographed. Restriction fragment length polymorphism (RFLP) profiles of the PCR amplicons were compared with the PCR amplicons of TnMERI1, Tn5085, and Tn5084 as references. The PCR product was analyzed by DNA sequencing.

2.3. 5’ Rapid Amplification of cDNA ends (50 RACE)

The transcription start sites of the ars operons were identified by using the 50-Full RACE Core Set (TaKaRa Bio Inc.). Bacillus sp. MB24 was inoculated at 1% (v/v) in 5 mL of LB broth and incubated at 37 ◦C with agitation for 3 h. NaAsO2 was added to 1 mL of culture to a final concentration of 5 mM, and the incubation was continued for another 15 min. Total RNA of Bacillus sp. MB24 was then extracted using the SV Total RNA Isolation System Kit (Promega, Madison, WI, USA) and reverse transcribed through the AMV Reverse transcriptase XL of the 50-Full RACE Core Set with primers RT-arsR1, RT-ORF3, and RT-arsR2. After removing the RNA by RNaseH, the produced cDNA was purified and self-cyclized by T4 RNA ligase. The cyclized cDNA was subjected to nested PCR with primer sets For-arsR1/Rev-arsR1, For-ORF3/Rev-ORF3, For-arsR2/Rev-arsR2. The primer sequences are shown in Table S1. The PCR product was analyzed by DNA sequencing.

2.4. Real-Time Quantitative Reverse Transcription-PCR (qRT-PCR) For real time qRT-PCR analysis, Bacillus sp. MB24 was inoculated into LB broth and incubated at 37 ◦C with agitation until the exponential phase. NaAsO2 was added to the cultures to a final concentration of 0, 1, 10, 100, and 1000 µM, and the incubation was continued for another 30 min. Total RNA of Bacillus sp. MB24 was then extracted using the SV Total RNA Isolation System Kit (Promega) and further treated with DNase I (TaKara Bio Inc.). Real-time qRT-PCR was performed using AccessQuick RT-PCR System (Promega) with SYBR green1 (Bio-Rad, Hercules, CA, USA) along with the Corbett Rotor-Gene 3000 (Qiagen, Hilden, Germany) under the following conditions: reverse transcription at 45 ◦C for 45 min, followed by 25 amplification cycles, each of which consisted of denaturation for 30 s at 94 ◦C, annealing for 30 s at 50 ◦C, extension for 1 min at 72 ◦C, and finally detecting the fluorescence at 78 ◦C for 15 s. The primer sets RTarsR1-F/R, RTorf3F/R, and RTarsR2F/R were used to amplify the transcript of arsR1, orf3, and arsR2, respectively. The primer sequences are shown in Table S1. Primer sets 16F and 16R were used to amplify the bacterial 16S rRNA gene for normalization. Every reaction was repeated at least five times. Microorganisms 2019, 7, 566 4 of 11

2.5. Genome Mining of Tn5084-like and TnARS1-like Transposon A total of 60,922 bacterial completed sequences/loci of Genbank bacterial division (December 2018) and whole genome sequences (4,178 strains) of Bacillus (August 2019) from NCBI (National Center for Biotechnology Information) were downloaded, and a selected whole genome sequence database was constructed. The complete sequences of transposon Tn5084 from Bacillus cereus RC607 (GenBank accession number: AB066362.1) and TnARS1 form Bacillus sp. MB24 (AY780525) were used as the query sequence against the constructed database for homologous cluster sequence similarity at the level of the entire gene cluster with a 30% sequence identify cut-off and 250 blastp hits mapped per query sequence using the MultiGeneBLAST program [28].

3. Results and Discussion

3.1. Identification of TnMARS1 in Bacillus sp. MB24 Among the 11 broad-spectrum mercury resistant Bacillus strains isolated from preserved samples of mercury-polluted Minamata Bay sediment, the mercury resistance determinant of Bacillus megaterium MB1 was the most studied [5,7,29]. In our previous study, the mercury resistance determinant, mer operon, of Bacillus sp. MB24 was identical to that of the MB1 strain [5], while the mercury resistance between MB1 and MB24 strains were different (M.-F. Chien, personal communication). Because the mer operon of B. megaterium MB1 is located in TnMERI1, a single primer PCR targeting the inverted-repeat (IR) sequence of TnMERI1 was employed to read the intact transposon among these Bacillus strains. Three strains, Bacillus cereus RC607 having Tn5084, B. megaterium MB1 having TnMERI1, and Bacillus sp. TW6 having Tn5085, were used as reference strains. Surprisingly, three types of fragments were detected (Figure1a). Amplicons from strains MB4, MB5, and MB6 were the same size as the Tn MERI1 PCR product (14.2-kbp). Amplicons from strains MB23, MB26, MB27, MB28, and MB29 were the same size as the Tn5084 and Tn5085 PCR product (11.5-kbp). However, amplicons from strains MB22, MB24, and MB25 were much longer (more than 25-kbp) than all three transposons (Figure1a). The amplicon from MB24 strains was sequenced, and the result showed that the mer operon and the transposable genes (tnpA, tnpR) of this amplicon are identical to that of TnMERI1 (M.-F. Chien, personal communication). However, this amplicon lacks the bacterial group II intron located between tnpR and merB3 in TnMERI1. Instead, a 13.8 kbp fragment was nested and resulted in a 25.2 kbp TnMERI1-like transposon. DNA sequencing of this 13.8 kbp fragment showed that it was flanked with a pair of 78-bp IR sequences, which had only one mismatched base. These IR sequences started with GGGG and ended with TAAG, which is common in the Tn3 family of transposons. At both outer ends of the IR, there were 5 bp of directed repeat (DR) sequences (TAGAA), which are known as the remains of transposition in class II transposons. A total of thirteen open reading frames (ORFs) were identified in this fragment through Blastx analysis in which rec and tnpA were classified into transposition-related genes, and others were classified into arsenic resistance related genes. This transposon-like fragment was then designated TnARS1, and the whole 26-kbp transposon in the MB24 strain was designated TnMARS1. The image of TnMARS1 is shown in Figure1b, in which Tn ARS1 was further analyzed. Microorganisms 2019, 7, x FOR PEER REVIEW 5 of 11 Microorganisms 2019, 7, 566 5 of 11

NN B S B H H BH

TnMERI1 (14.2 kb) res Bacillus megaterium MB1 mer determinants group II intron tnpA IR tnpR IR

14.2 kb PCR products (MB4, MB5, MB6)

Tn5084 (11.5 kb) res (Bacillus cereus RC607) mer determinants tnpA IRtnpR IR

11.5 kb PCR products (MB23, MB26, MB27, MB28, MB29) H Tn5085 (11.5 kb) res (Bacillus sp. TW6) mer determinants tnpA IRtnpR IR

26 kb PCR products (MB22, MB24, MB25)

BH HB H

14 kp

1 kb

(a)

TnARS1 (13.8 kb)

arsB arsC orf11 arsR1 orf5 arsD orf12 recorf3 orf4 arsR2 arsA tnpA tnpR tnpA res mer operon IR DRIR IR DR IR

(b)

Figure 1. a Figure 1.( )(a The) The restriction restriction fragment fragment length length profile profile of amplified of amplified PCR amplicons PCR amplicons from mercury from resistantmercury Bacillus strains; (b) schematic representation of putative TnMARS1. Hatched boxes indicate the resistant Bacillus strains; (b) schematic representation of putative TnMARS1. Hatched boxes indicate sequenced region. the sequenced region. 3.2. Characterization of TnARS1 in Bacillus sp. MB24. 3.2. Characterization of TnARS1 in Bacillus sp. MB24. 3.2.1. Sequence Analysis of TnARS1 3.2.1. Sequence Analysis of TnARS1 The sequence of TnARS1 was furthered analyzed. Translated amino acids of rec in TnARS1 showedThe high sequence similarity of Tn toARS recombinases.1 was furthered The closest analyzed. one isTranslated a recombinase amino from acidsBacillus of rec wiedmanniiin TnARS1 (accessionshowed high no. similarity PTC10770.1), to recombinases which shares. The 97% closest identity. one Moreover, is a recombinase the sequence from thatBacillus is needed wiedmannii for resolvase(accession (a no. kind PTC10770.1), of recombinase) which binding shares during97% identity. recombination Moreover, was the also sequence recognized that upstreamis needed offor recresolvasein TnARS1 (a kind. On of the recombinase) other hand, binding the translated during recombination amino acid sequence was also of recognizedtnpA in Tn ARSupstream1 showed of rec highin Tn similarityARS1. On to the transposases. other hand, The the closest translated one isamino a transposase acid sequence from Bacillusof tnpAstratosphericus in TnARS1 showed(accession high no.similarity KML06687.1), to transposases. which shares The cl 96%osest identity. one is a The transposase TnpA in from TnARS Bacillus1 was stratosphericus classified under (accession the family no. ofKML06687.1), transposase Tnwhich3 based shares on 96% its conserved identity. The domain, TnpA DDEin Tn motif,ARS1 was which classified is necessary under in the catalyzing family of transposition.transposase Tn These3 based results, on includingits conserved the DR domain,/IR sequence DDE describedmotif, which in 3.1 is, suggested necessary an in occurrence catalyzing oftransposition. a transposition These event results, by Tn includingARS1, which the DR/IR resulted sequence in the Tn describedMARS1. in However, 3.1, suggested the mismatch an occurrence of IR, whichof a transposition might have happenedevent by Tn duringARS1, transposition, which resulted made in the Tn ARSTnMARS1 no longer1. However, effective the as mismatch a transposon. of IR, whichAccording might have to happened the results during of Blastp, transposition, the arsenic-resistance made TnARS1 no genes longer were effective divided as a transposon. into two geneAccording clusters ( arsto theoperon) results leadof Blastp, by twothe arsenic-resistance regulatory genes genes (arsR were), arsR1-orf3-orf4-orf5 divided into two andgene arsR2-arsB-arsC-arsD-arsA-orf11-orf12clusters (ars operon) lead by two regulatory. The arsR1 genesand (arsRarsR2), arsR1-orf3-orf4-orf5in TnARS1 were identified and arsR2-arsB-arsC- as arsenic resistancearsD-arsA-orf11-orf12 operon repressor. The genes.arsR1 Theand translatedarsR2 in Tn aminoARS1 acids were of identifiedarsR1 and arsR2as arsenicpresented resistance a conserved operon metal-bindingrepressor genes. motif, The translated EXCVC(D amino/E)L, and acids the of DNAarsR1 and binding arsR2 helix-turn-helix presented a conserved motif as metal-binding with other motif, EXCVC(D/E)L, and the DNA binding helix-turn-helix motif as with other reported ArsR

Microorganisms 2019, 7, 566 6 of 11 Microorganisms 2019, 7, x FOR PEER REVIEW 6 of 11 reported ArsR proteins. The Blastp of ORF3-ORF4-ORF5 did not show similarity with any protein proteins. The Blastp of ORF3-ORF4-ORF5 did not show similarity with any protein related to arsenic related to arsenic resistance. The conserved domains of ORF3 showed high similarity with glutamate resistance. The conserved domains of ORF3 showed high similarity with glutamate synthase. The synthase. The conserved domains of ORF4 showed high similarity with a flavoprotein monooxygenase. conserved domains of ORF4 showed high similarity with a flavoprotein monooxygenase. ORF5 was ORF5 was identified as a GNAT family N-acetyltransferase. The alignment result of the gene cluster identified as a GNAT family N-acetyltransferase. The alignment result of the gene cluster arsR2-arsB- arsR2-arsB-arsC-arsD-arsA-orf11-orf12 showed that the former five genes are typical five-gene type arsC-arsD-arsA-orf11-orf12 showed that the former five genes are typical five-gene type arsenic arsenic resistance genes arsRBCDA, which are usually found in plasmids of gram-negative bacterium, resistance genes arsRBCDA, which are usually found in plasmids of gram-negative bacterium, in in which there is no orf11 and orf12. On the other hand, the Blastp results showed similarity to the which there is no orf11 and orf12. On the other hand, the Blastp results showed similarity to the chromosomal arsenic resistance operon of Bacillus cereus Rock1-3, in which ORF11 and ORF12 were also chromosomal arsenic resistance operon of Bacillus cereus Rock1-3, in which ORF11 and ORF12 were found as a adhesin and protein phosphatase, respectively. The Blastp result of each gene in TnARS1 also found as a adhesin and protein phosphatase, respectively. The Blastp result of each gene in is shown in Table S2. These data suggested that TnARS1, a 14-kbp unique class II transposon, was TnARS1 is shown in Table S2. These data suggested that TnARS1, a 14-kbp unique class II transposon, inserted into Tn5085 to form TnMARS1. The sequence of TnARS1 was deposited into GeneBank with was inserted into Tn5085 to form TnMARS1. The sequence of TnARS1 was deposited into GeneBank accession number AY780525. with accession number AY780525. 3.2.2. Transcription of ars Operons in Bacillus sp. MB24 3.2.2. Transcription of ars Operons in Bacillus sp. MB24 GENETYX-MAC (GENETYX Co.) was employed to predict the potential promoter regions of GENETYX-MAC (GENETYX Co.) was employed to predict the potential promoter regions of the two ars operons. There were three putative regions: one was upstream arsR1, one was upstream the two ars operons. There were three putative regions: one was upstream arsR1, one was upstream orf3, and another was upstream arsR2. In order to determine the transcription start sites of two ars orf3, and another was upstream arsR2. In order to determine the transcription start sites of two ars mRNA, total RNA of Bacillus sp. MB24 were extracted after inducing by arsenite and were subjected to mRNA, total RNA of Bacillus sp. MB24 were extracted after inducing by arsenite and were subjected 50 RACE. The 50 terminus of arsR1 and arsR2 mRNA were precisely identified to be 30 nt and 97 nt to 5′ RACE. The 5′ terminus of arsR1 and arsR2 mRNA were precisely identified to be 30 nt and 97 nt upstream of the initiation codon of ArsR1 and ArsR2, respectively. These results demonstrated that upstream of the initiation codon of ArsR1 and ArsR2, respectively. These results demonstrated that there are two operons, ars1 operon and ars2 operon, in TnARS1. Moreover, no 50 RACE amplicon was there are two operons, ars1 operon and ars2 operon, in TnARS1. Moreover, no 5′ RACE amplicon was obtained upstream of orf3, which suggested a consensus result that orf3 belongs to the same operon obtained upstream of orf3, which suggested a consensus result that orf3 belongs to the same operon as arsR1 and thus is transcribed along with arsR1. The putative promoter regions (-35, -10) and the as arsR1 and thus is transcribed along with arsR1. The putative promoter regions (-35, -10) and the transcription start point (+1) of arsR1 and arsR2 are shown in Figure2. The response of ars1 and ars2 transcription start point (+1) of arsR1 and arsR2 are shown in Figure 2. The response of ars1 and ars2 to different concentrations of arsenic was further investigated by quantitative real time RT-PCR. In to different concentrations of arsenic was further investigated by quantitative real time RT-PCR. In the absence of arsenite, transcription of arsR1 and arsR2 presented basal levels of 0.1 and 0.06 fold of the absence of arsenite, transcription of arsR1 and arsR2 presented basal levels of 0.1 and 0.06 fold of 16S rRNA transcripts, respectively. The transcription response of the two arsR genes responded to 16S rRNA transcripts, respectively. The transcription response of the two arsR genes responded to different concentrations of arsenite as shown in Figure3. One micromolar of arsenite was su fficient to different concentrations of arsenite as shown in Figure 3. One micromolar of arsenite was sufficient induce a more than 4-fold increase of arsR2 transcription, while the same response of arsR1 needed to induce a more than 4-fold increase of arsR2 transcription, while the same response of arsR1 needed 100 µM of arsenite. The transcription induction of both arsR genes was positively correlated with the 100 μM of arsenite. The transcription induction of both arsR genes was positively correlated with the concentration of arsenite. In terms of sensitivity, arsR2 was observed to increase up to 18-fold of the concentration of arsenite. In terms of sensitivity, arsR2 was observed to increase up to 18-fold of the basal level, while arsR1 only increased by 4-fold. These results showed that the arsR2 operon reacts to basal level, while arsR1 only increased by 4-fold. These results showed that the arsR2 operon reacts arsenite more sensitively and strikingly than arsR1 does. In addition, arsR1 and orf3 showed coherent to arsenite more sensitively and strikingly than arsR1 does. In addition, arsR1 and orf3 showed expression profiles suggesting that they belong to the same operon. coherent expression profiles suggesting that they belong to the same operon. (a) TTTCAACGTT TCATAGACAA TACGTAAATG TTTGTAAAGG TATACCTTTT TAAACTAAAT

-35 GCTTTTTACA ACTAATAAAT CAATTTTTTT TGATTTATTA GTTGTAAAAG TAACGAAAGA

-10

CGTATATATT ATAAATATCA AAAAAAATTG ATGAAAGGGA ATTACCATGA AAGAAATTAA +1 arsR1

Figure 2. Cont.

Microorganisms 2019, 7, x FOR PEER REVIEW 7 of 11 Microorganisms 2019, 7, 566 7 of 11 Microorganisms 2019, 7, x FOR PEER REVIEW 7 of 11

(b) -35 -10 (b) -35 -10 AAGATTCTGT AAAGGTGAGA GTTTTCAGTT GAACATATAA GCAAATAGTT ATATACTTAT AAGATTCTGT AAAGGTGAGA GTTTTCAGTT GAACATATAA GCAAATAGTT ATATACTTAT

TTTGAAAGAG AGAATAAAGT AACTTAAAAG AATAATTCAC TTTATTTTTA CACAACATAT TTTGAAAGAG+1 AGAATAAAGT AACTTAAAAG AATAATTCAC TTTATTTTTA CACAACATAT +1 AAGCATATGA TTATGTAATT ATAACAAGGT AGGTGTAGAT ATGGAAAAAA CAGTTGTAGA arsR2 AAGCATATGA TTATGTAATT ATAACAAGGT AGGTGTAGAT ATGGAAAAAA CAGTTGTAGA arsR2

Figure 2. Putative promoter regions (-35, -10) and the transcription start point (+1) of (a) arsR1 and (b) Figure 2. Putative promoter regions (-35, -10) and the transcription start point (+1) of (a) arsR1 and arsR2Figure. The 2. Putative arrows showedpromoter the regions transcrip (-35,tion -10) start and sites the transcription(+1), and the coloredstart point words (+1) showedof (a) arsR1 the andarea (ofb) (b) arsR2. The arrows showed the transcription start sites (+1), and the colored words showed the area arsR1arsR2 .and The arsR2 arrows, respectively. showed the transcription start sites (+1), and the colored words showed the area of of arsR1 and arsR2, respectively. arsR1 and arsR2, respectively. 10 10

1 arsR1 1 orf3arsR1 arsR2orf3 arsR2 0.1 gene/16S rRNA) 0.1 gene/16S rRNA) relativeexpression ratio (target 0.01 relativeexpression ratio (target 0.01 0 1 10 100 1000 As(III) μM 0 1 10 100 1000 As(III) μM Figure 3. Relative transcription of ars operons under different concentrations of arsenite. Figure 3. Relative transcription of ars operons under different concentrations of arsenite. 3.2.3. ArsenicFigure Resistance 3. Relative Conferred transcription by ars ofOperon ars operons in Bacillus under different subtilis concentrations168 of arsenite. 3.2.3. Arsenic Resistance Conferred by ars Operon in Bacillus subtilis 168 3.2.3.The Arsenic function Resistance of the ars1 Conferredand ars2 operonby ars Operon as an arsenic in Bacillus resistance subtilis operon 168 was further investigated. FragmentsThe function containing of the the ars1ars1 andoperon, ars2 operonars2 operon as anand arsenic both resistance operons wereoperon cloned was further into Bacillus investigated. subtilis The function of the ars1 and ars2 operon as an arsenic resistance operon was further investigated. 168Fragments∆arsB, which containing is a strain the withars1 operon, a mutated ars2arsB operonin its and genome. both operons The minimum were cloned inhibition into concentrationBacillus subtilis Fragments containing the ars1 operon, ars2 operon and both operons were cloned into Bacillus subtilis (MIC)168ΔarsB and, maximumwhich is a strain tolerance with concentration a mutated arsB (MTC) in its ofgenome. the transformed The minimu strainsm inhibition against concentration arsenite and 168ΔarsB, which is a strain with a mutated arsB in its genome. The minimum inhibition concentration arsenate(MIC) and were maximum investigated. tolerance The results concentration displayed (MTC) the loss of the of function transformed of arsB strains, which against resulted arsenite in 2-fold and (MIC) and maximum tolerance concentration (MTC) of the transformed strains against arsenite and andarsenate 19-fold were reductions investigated. in the MTCsThe result of arsenites displayed and arsenate,the loss of respectively. function of MICarsB, alsowhich exhibited resulted the in same2-fold arsenate were investigated. The results displayed the loss of function of arsB, which resulted in 2-fold trendand 19-fold (Table1 ).reductions The reduction in the was MTCs only of slightlyarsenite complemented and arsenate, respectively. when introducing MIC also the ars1exhibitedoperon, the and 19-fold reductions in the MTCs of arsenite and arsenate, respectively. MIC also exhibited the butsame the trend arsenate (Table and arsenite1). The resistancereduction waswas significantly only slightly recovered complemented by introducing when introducing the ars2 operon the into ars1 same trend (Table 1). The reduction was only slightly complemented when introducing the ars1 theoperon, host bacillus. but the arsenate All of these and results arsenite revealed resistance that wasars2 significantlyoperon is a functional recovered operon. by introducing the ars2 operonoperon, into but thethe hostarsenate bacillus. and Alarsenitel of these resistance results revealedwas significantly that ars2 recoveredoperon is aby functional introducing operon. the ars2 operon into the host bacillus.Table 1. AlComplementaryl of these results effect revealed of the ars1 that and ars2 ars2 operon operon. is a functional operon. Table 1. Complementary effect of the ars1 and ars2 operon. Table 1. ComplementaryArsenic Resistance effect of the (mM) ars1 and ars2 operon. Arsenic ResistanceAs(III) (mM) As(V) Arsenic Resistance (mM) MTCAs(III) MIC MTC MICAs(V) As(III) As(V) Bacillus subtilis 168 MTC 4MIC 6 38MTC 40 MIC Bacillus subtilisBacillus subtilis168 168∆arsB MTC4 2 MIC 46 2MTC38 4 MIC40 BacillusBacillusBacillus subtilis subtilis subtilis 168 168Δ168arsB∆ arsB /pHYARS124 2 446 6382 8 404 BacillusBacillus subtilis subtilis 168Δ168arsB∆arsB /pHYARS22 4 64 202 30 4 Bacillus subtilisBacillus 168Δ subtilisarsB/pHYARS1168∆arsB/pHYARS3 2 14 16 4 10 6 20 8 BacillusBacillus subtilissubtilis 168168ΔΔBacillusarsBarsB/pHYARS2/pHYARS1sp. MB24 42 12 146 4 2820 6 30 30 8 BacillusBacillus subtilissubtilis 168168ΔΔarsBarsB/pHYARS3/pHYARS2 144 166 1020 2030 3.3. GenomeBacillus Mining subtilisBacillus of 168 Tnsp.Δ5084 MB24arsB-like/pHYARS3 and TnARS 1-like1214 Regions 1416 2810 3020 Bacillus sp. MB24 12 14 28 30 There are 11 broad-spectrum mercury resistant Bacillus strains isolated from mercury-polluted 3.3. Genome Mining of Tn5084-like and TnARS1-like Regions Minamata3.3. Genome Bay Mining sediment of Tn [55084]. Among-like and these, TnARSBacillus1-like megaterium Regions MB1 harbors one group II intron region

Microorganisms 2019, 7, 566 8 of 11

Microorganisms 2019, 7, x FOR PEER REVIEW 8 of 11 in the Tn5084Theretransposon are 11 broad-spectrum [30]. In this mercury study, we resistant found thatBacillusBacillus strainssp. isolated strains from MB4, mercury-polluted MB5, MB6 have the sameMinamata transposon Bay as sediment MB1. Further, [5]. Among we found these, that BacillusBacillus megateriumsp. strains MB1 MB22, harbors MB24, one MB25 group also II intron harbor a Tn5084region-like in mercury the Tn5084 resistance transposon operon [30]. transposon In this study, nested we found by an that arsenic Bacillus resistance sp. strains transposon MB4, MB5, (Tn MB6ARS1 ) (Figurehave1). the It is same intriguing transposon that as both MB1. these Further, two mobilewe found elements that Bacillus are locatedsp. strains between MB22, MB24, the res MB25site andalso the mer operon.harbor Thesea Tn5084 results-like suggestedmercury resistance that this region operon might transposon be a “hot nested spot” forby mobilean arsenic elements resistance to jump in. Intransposon order to survey (TnARS1 this) (Figure phenomenon, 1). It is weintriguing used Tn that5084 bothas thethese gene two cluster mobile query elements against are alocated bacterial databasebetween (BCT the GenBank res site and bacterial the mer division)operon. These and results the whole suggested genome that sequence this region database might be of a Bacillus“hot spot”(4178 wholefor genome mobile sequences).elements to jump The resultsin. In orde showedr to survey that there this arephenomenon, some Tn5084 we-like used transposons Tn5084 as the in geneBacillus sp. strainscluster (Figure query against4a). However, a bacterial there database are no (BCT transposons GenBank or bacterial mobile elementsdivision) nestedand the in whole the Tn genome5084-like sequence database of Bacillus (4178 whole genome sequences). The results showed that there are some transposon from Bacillus sp. except TnMERI1 and TnMARS1. On the other hand, we used the TnARS1 Tn5084-like transposons in Bacillus sp. strains (Figure 4a). However, there are no transposons or transposon as a query for multi-gene cluster analysis. The results showed that some Bacillus sp. strains mobile elements nested in the Tn5084-like transposon from Bacillus sp. except TnMERI1 and containTnMARS a gene1. clusterOn the similarother hand, to MB24, we used (ars1 theoperon TnARS1 and transposonars2 operon), as a and query some forBacillus multi-genestrains cluster harbor onlyanalysis. the ars2 operonThe results (Figure showed4b). However,that some noBacillus arsenic sp. operon strains wascontain identified a gene incluster a transposon, similar toincluding MB24, any mercury(ars1 operon resistance and ars2 transposon. operon), and some Bacillus strains harbor only the ars2 operon (Figure 4b). However,The increase no inarsenic horizontal operon gene was transferidentified due in toa environmentaltransposon, including stresses any has mercury been demonstrated. resistance Environmentaltransposon. stresses enhance the activity of transposable elements for extensive genomic changes that facilitateThe increase the adaptation in horizontal of populationsgene transfer [due31]. to Theenvironmental arsenic resistant stresses operonhas been (demonstrated.ars1 and/or ars2 operon)Environmental were also found stresses in enhanceBacillus thegenomes activity from of transposable different sources, elements such for extensive as rocks, genomic landfill, changes fermented bacterialthat facilitate culture andthe adaptation patients [ 32of– 34populations]. TnMERI [31].1-like The transposonsarsenic resistant were operon also present(ars1 and/or in di ars2fferent sources,operon) such were as also mouse found guts, in Bacillus water, genomes beef salad, from anddifferent canned sources, chocolate such as beveragerocks, landfill, [7]. fermented These toxic bacterial culture and patients [32–34]. TnMERI1-like transposons were also present in different metal/metalloid resistance operons containing transposons were found in various environments, not sources, such as mouse guts, water, beef salad, and canned chocolate beverage [7]. These toxic only in contaminated sites. However, to the best of our knowledge, TnMRS1 and TnMERI1 are the metal/metalloid resistance operons containing transposons were found in various environments, not onlyonly samples in contaminated that an arsenic sites. resistance However, transposon to the best andof our a group knowledge, II intron TnMRS nested1 and in a Tn mercuryMERI1 are resistance the transposon,only samples respectively. that an As arsenic they areresistance all identified transposon from Minamataand a group Bay, II aintron site famous nested forin man-induceda mercury mercuryresistance contamination, transposon, anthropogenicrespectively. As pollutionthey are all might identified be the from main Minamata driving Bay, force a site for famous evolution for of the organisms.man-induced The mercury new findings contamination, of this studyanthropogenic might be pollution the result might of a be horizontal the main genedriving transfer force for event drivenevolution by environmental of the organisms. stresses. The Thenew presencefindings of of this Tn MARSstudy might1 found be the in Minamata result of a horizontal Bay also suggests gene investigatingtransfer event other driven mercury by environmental contaminated stresses. sites, which The presence might reveal of TnMARS further1 found diversity in Minamata and structure Bay of mercuryalso resistancesuggests investigating transposons. other mercury contaminated sites, which might reveal further diversity and structure of mercury resistance transposons.

(a)

Figure 4. Cont. Microorganisms 2019, 7, 566 9 of 11 Microorganisms 2019, 7, x FOR PEER REVIEW 9 of 11

Bacillus sp. MB24 (TnARS1, AY780525) ars1 operon ars2 operon 1000 bp rec tnpA

Bacillus cereus Rock1-3 (CM000728)

Bacillus thuringiensis BM-BT15426 (CP020723)

Bacillus sp. HBCD-sjtu (CP025122)

Bacillus cereus Rock1-15 (CM000729)

Transcription regulator/ Arsenical resistance Bacillus cereus AH820 operon repressor (DQ889677)

(b)

FigureFigure 4. Selected 4. Selected blasting blasting results results of of (a )(a Tn)Tn50845084-like-like and and ((bb))Tn TnARSARS1-like1-like transposons transposons among among bacteria bacteria by usingby using the MultiGeneBlast the MultiGeneBlast analysis. analysis. The databasesThe databases are the are BTC the database BTC database (Genbank: (Genbank: bacterial bacterial division, Decemberdivision, 2018) December and the 2018) whole and the genome whole sequencesgenome sequences of the Bacillus of the Bacillusdatabase database (NCBI, (NCBI, August August 2019). The threshold2019). The ofthreshold gene identity of gene is identity 30% and is the30% gene and clusters.the gene tnpAclusters.: transposase tnpA: transposase gene; tnpR gene;: resolvase tnpR: gene;resolvaseB3: organomercurial gene; B3: organomercurial lyase gene merB3 lyase; R gene: mercury-responsive merB3; R: mercury-responsive transcriptional transcriptional regulator gene merRregulator; E: mercury gene resistance merR; E: genemercurymerE resistance; T: mercury gene transpor merE; T: gene mercurymerT transpor; P: metal gene binding merT protein; P: metal gene merP;bindingA: Mercuric protein reductase gene merP gene; A: MercuricmerA; R2 reductase: mercury-responsive gene merA; R2: transcriptional mercury-responsive regulator transcriptional gene merR2 ; B2: organomercurialregulator gene merR2 lyase; B2 gene: organomercurialmerB2; B1: organomercurial lyase gene merB2; B1 lyase: organomercurial gene merB1. GenBanklyase gene accessionmerB1. numbersGenBank are listed accession following numbers bacterial are listed strains. following TnMERI1 bacterial: LC152290; strains. Tn 5084TnMERI1: AB066362.1;: LC152290;Bacillus Tn5084 cereus: #17: JYFW01000036;AB066362.1; BacillusB. cereus cereus PE8-121b:#17: JYFW01000036; LRPI01000016.1. B. cereus PE8-121b: LRPI01000016.1.

4. Conclusions4. Conclusions The resultsThe results of this of this study study reveal reveal that that an an arsenic arsenic resistance resistance transposon, Tn TnARSARS1, 1,was was nested nested in a in a Tn5084Tn-like5084-like mercury mercury resistance resistance transposon, transposon, designated designated as Tn asMARS TnMARS1. Tn1.ARS Tn1ARS contains1 contains two arstwooperons, ars operons, in which the ars2 operon is more functional. Results of genome mining showed that TnARS1 in which the ars2 operon is more functional. Results of genome mining showed that TnARS1 is the first is the first identified arsenic resistance transposon, and TnMARS1 is the first arsenic resistance identified arsenic resistance transposon, and TnMARS1 is the first arsenic resistance transposon nested transposon nested in another transposon. These results suggest that a genetic element recruitment in another transposon. These results suggest that a genetic element recruitment event has occurred event has occurred through TnMERI1-like transposons in Minamata Bay, which might be a strategy throughfor Bacilli TnMERI to survive1-like transposonsin this severe inenvironment. Minamata Bay, which might be a strategy for Bacilli to survive in this severe environment. Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Table S1: Primers used Supplementaryin this study, Materials: Table S2 GeneThe analysis following of TnARS1 are available based on online Blastp. at http://www.mdpi.com/2076-2607/7/11/566/s1, Table S1: Primers used in this study, Table S2: Gene analysis of TnARS1 based on Blastp. Author Contributions: M.-F.C.; data curation, methodology, investigation, project administration, validation, Authorvisualization, Contributions: writingM.-F.C.; - original data draft curation, preparation, methodology, writing- review investigation, & editing, funding project administration,acquisition, Y.-N.H.; validation, data visualization,curation, methodology, writing—original investigation, draft preparation, validation, visualization, writing—review writing & editing,- originalfunding draft preparation, acquisition, writing- Y.-N.H.; datareview curation, & editing, methodology, H.-E.Y.; investigation,data curation, validation,investigation, visualization, M.N.; methodology, writing—original data curation, draft investigation, preparation, writing—reviewvalidation K.M; & editing, methodology, H.-E.Y.; data data curation, curation, investigation, investigation, validation, M.N.;methodology, G.E, and C.-C.H.; data project curation, administration, investigation, validationwriting K.M.; - review methodology, & editing, supervision, data curation, funding investigation, acquisition. validation, G.E., and C.-C.H.; project administration, writing—review & editing, supervision, funding acquisition. Funding: This research was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Funding: This research was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Number 19H02654 (Grant-in-Aid for Scientific Research(B)). Part of this research was supported by (JSPS) Grant Number 19H02654 (Grant-in-Aid for Scientific Research(B)). Part of this research was supported by (JSPS) KAKENHIKAKENHI Grant Grant Number Number 18F18393 18F18393 (Grant-in-Aid (Grant-in-Aid for for JSPS JSPS Research Research Fellow).

Acknowledgments: We appreciate Fan-Fan Chen from National Chung Hsing University, Taichung, Taiwan and Kohei Obata from Tohoku Gakuin University, Miyagi, Japan for their experimental support in this study. Microorganisms 2019, 7, 566 10 of 11

Conflicts of Interest: The authors declare no conflict of interest.

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